1. Field of the Invention
The invention relates to the field of cellular electroporation for gene, protein, or drug therapy and in particular to the application of gene-therapy for the rejection of heart or organ transplantation, cardiovascular disease and cancer in any organ.
2. Description of the Prior Art
Electroporation is a technique involving the application of short duration, high intensity electric field pulses to cells or tissue. The electrical stimulus causes membrane destabilization and the subsequent formation of nanometersized pores. In this permeabilized state, the membrane can allow-passage of DNA, enzymes, antibodies and other macromolecules into the cell. Electroporation holds potential not only in gene therapy, but also in other areas such as transdermal drug delivery and enhanced chemotherapy. Since the early 1980s, electroporation has been used as a research tool for introducing DNA, RNA, proteins, other macromolecules, liposomes, latex beads, or whole virus particles into living cells. Electroporation efficiently introduces foreign genes into living cells, but the use of this technique had been restricted to suspensions of cultured cells only, since the electric pulse are administered with cuvette type electrodes.
Electroporation is commonly used for in vitro gene transfection of cell lines and primary cultures, but limited work has been reported in tissue. In one study, electroporation-mediated gene transfer was demonstrated in rat brain tumor tissue. Plasmid DNA was injected intra-arterially immediately following electroporation of the tissue. Three days-after shock treatment expression of the lacZ gene or the human monocyte chemoattractant protein-1 (MCP-1) gene was detected in electroporated tumor tissue between the two electrodes, but not in adjacent tissue. Electroporation has also been used as a tissue-targeted method of gene delivery in rat liver tissue. This study showed that the transfer of genetic markers xcex2-glactosidase (xcex2-gal) and luciferase resulted in maximal expression at 48 hr, with about 30-40% of the electroporated cells expressing xcex2-gal, and luciferase activities reaching peak levels of about 2500 xcexcg/mg of tissue.
In another study, electroporation of early chicken embryos was compared to two other transfection methods: microparticle bombardment and lipovection. Of the three transfection techniques, electroporation yielded the strongest intensity of gene expression and extended to the largest area of the embryo. Most recently, an electroporation catheter has been used for delivery heparin to the rabbit arterial wall, and significantly increased the drug delivery efficiency.
Electric pulses with moderate electric field intensity can cause temporary cell membrane permeabilization (cell discharge), which may then lead to rapid genetic transformation and manipulation in wide variety of cell types including bacteria, yeasts, animal and human cells, and so forth. On the other hand, electric pulses with high electric field intensity can cause permanent cell membrane breakdown (cell lysis). According all the knowledge available now, the voltage applied to any tissue must be as high as 100-200 V/cm. if it is to be used on large animal or a human organ, such as human heart, it must be several kV. It will cause enormous tissue damage. Therefore, this technique is still not applicable for clinical use.
Electroporation is commonly used for in vitro gene transfection of cell lines and primary cultures, but limited work has been reported in organized tissue. According to the theory in in vitro study, the membrane voltage, Vm, at different loci on phospholipid bilayer spheres during exposure in a homogenous electric field of duration t, can be calculated from
Vm=1.5 rc E cos xcex1[1xe2x88x92exp(xe2x88x92t/T)]xe2x80x83xe2x80x83(1)
where E is the electric field strength, rcis the cell radius, xcex1 is the angle relative to the direction of the electric field, and T is the capacitive-resistive time constant. Pore-formation will result at those spherical coordinates exposed to a maximal potential difference, which is at the poles facing the electrodes (cos xcex1=1 for xcex1=0; cos xcex1=xe2x88x921 for xcex1=TT). Generally, electric field strengths on the order of 1 to 1.5 kV/cm for duration of a few xcexcs to a few ms are sufficient to cause transient permeabilization in 10-xcexcm outer-diameter spherical cells. A recent study shows that isolated mitochondria, because of their correspondingly smaller size, require 7- to 10-fold higher electric field strengths to incorporate a 7.2-kilobase plasmid DNA. Mitochondrial outer-membrane fusion at lower electric field strengths of=2.5 kV/cm also has been observed.
According to three most recent studies of gene delivery to rat liver and embryonic chick heart, the voltage applied to any tissue must be as high as 100-200 V/cm. However, this is much lower than the theoretical level calibrated from equation (1). For gene delivery in an organ the magnitude of voltage can be much lower than that for cell suspension. However, more accurate calibration is not available. Even according this prior art information, if one wishes to use electroporation on a large animal or human organ, such as human heart, it must be at lest several kV. Such voltage levels will cause enormous tissue damage. Therefore, this technique is still not applicable for use in any large animal or human organs.
What is needed is some means of increasing electroporation without causing tissue damage.
The invention is an apparatus for electroporation of biological cells. The invention comprises a perfusion medium in which the biological cells are disposed, typically in the form of organized tissue or a whole organ. The tissue or organ may be either in vivo or ex vivo. A source of a low voltage, pulsed, DC electric gradient field is established across the biological cells, tissue or organ. A source of genes, proteins, and/or drugs to be delivered through perfusion of the organ, then be transferred into and from the perfusion medium into the biological cells is also provided by conventional means.
The source of a low voltage, pulsed, DC electric gradient field is a conductive array disposable in proximity or contact to the biological cells, tissue or organ. In the case where the tissue or organ has a cavity the array is disposed in and conformable to the interior walls of the cavity. In the case of a solid organ, the array is conformable to the external wall of the organ. In the case where the tissue or organ is ex vivo the array is in contact with an exterior surface of the tissue or organ.
In one embodiment the conductive array may be comprised of a first part disposable in contact with a cavity defined in the tissue or organ, and a second part disposable in contact with the exterior surface of the tissue or organ. Opposite polarities of voltage are applied to the first and second parts of the conductive array. The conductive array is comprised of a flexible mesh conformable to the cavity.
In another embodiment the conductive array is comprised of a first plurality of electrodes having a first polarity and a second plurality of electrodes having a second polarity. The low voltage, pulsed, DC electric gradient field is established between the first plurality of electrodes and the second plurality of electrodes.
The source of a low voltage, pulsed, DC electric gradient field provides a high frequency pulsed DC field applied to the biological cells at a group repetition rate. Preferably the low voltage, pulsed, DC electric gradient field provides a voltage gradient across the biological cells of 0.1 to 10 V/cm. In the illustrated embodiment the source of a low voltage, pulsed, DC electric gradient field provides a pulse of DC voltage of approximately 1-5 ms long at approximately 500 Hz with a 50% duty cycle. The low voltage, pulsed, DC electric gradient field is provided in a burst of pulses of DC voltage followed by a rest period of the order of several seconds to 10 minutes, typically of the order of 5 seconds. There are typically 5 to 10 short pulses in every burst.
The apparatus further comprises an inflatable balloon disposed within the conductive array.
In another embodiment the conductive array is a pair of opposed plates between which the biological cells, tissue or organ are disposed. In the case where the tissue defines a lumen, the conductive array is a cylindrical mesh inflatable within the lumen. The cylindrical mesh is comprised of a plurality of separate electrodes provided in two sets with opposing polarities.
For a solid organ, such as the prostate, liver, and kidney, ex vivo, the gene, protein and drug delivered to the organ through an artery or vein, then a conductive array mesh in contact with the organ provides the organ with negative charge, and a central wire will be placed in the main artery is provided with a positive charge. The burst pulses of DC voltage are then applied to the mesh.
The invention is also defined as a method for electroporation of biological cells comprising the steps of disposing the biological cells, tissue or organs in a perfusion medium, and establishing a low voltage, pulsed, DC electric gradient field across the biological cells. Genes, proteins, and/or drugs are transferred into and then from the perfusion medium into the biological cells, tissue or organs. As discussed above the low voltage, pulsed, DC electric gradient field is applied as a high frequency pulsed DC field to the biological cells, tissue or organs at a group repetition rate. The low voltage, pulsed, DC electric gradient field is applied as a voltage gradient across the biological cells of 0.1 to 10 V/cm. For example, in one embodiment it is applied as a pulse of DC voltage of approximately 1 ms long pulse at approximately 500 Hz with approximately a 50% duty cycle. The low voltage, pulsed, DC electric gradient field is applied as a burst of pulses of DC voltage followed by a rest period of the order of several seconds to 10 minutes. The burst of pulses of DC voltage is applied as a burst of pulses of the order of 0.1-2 seconds in duration.
The invention now having been briefly summarized, it is illustrated in the following drawings wherein like elements are referenced by like numerals.